The goal of this study is to develop a rapid, sensitive method for analysis of estrogen metabolites in a small volume of plasma, for use in large-scale epidemiological studies of cancer risk. In addition to the major endogenous estrogens, specific estrogen metabolites such as 16 alpha-hydroxyestrone, 4-hydroxyestrone and 4-hydroxyestradiol have been hypothesized to play important roles in the etiology of cancer. Despite the potential significance of these compounds in carcinogenesis, blood levels have not yet been evaluated in epidemiological studies. In fact, no methods are available which can measure the extremely low concentrations at which these compounds are likely present in blood. Gas Chromatography/Mass Spectrometry (GC/MS) has better specificity than immunoassay methods for the analysis of estrogens, and stable isotope-labelled internal standards can be used to correct for recovery during analysis. For these reasons, GC/MS has become the "gold standard" for the measurement of sex steroids in biological samples such as urine. However, current GC/MS methods lack sufficient sensitivity for the measurement of the low levels found in blood. Negative Ion Chemical Ionization Mass Spectrometry (NCI-MS) of halogenated, electron-capturing derivatives has been used successfully to measure picogram quantities of compounds such as aromatic amines in biological fluids, and similar derivatives can be prepared for estrogens. We propose to develop and validate a sensitive and specific GC/NCI-MS method for analysis of estrogen metabolites in blood. Pre-purification of samples by previously developed high performance liquid chromatography (HPLC) methods will also improve detection limits. Analysis of blood samples from postmenopausal women, where estrogen levels are very low, will validate the sensitivity and precision of the method. Development of a rapid and sensitive method for the measurement of estrogen metabolites in a small volume of plasma will enable epidemiologists to perform prospective studies on the associations between estrogen metabolites and cancer risk.